DNA vaccines, the third generation of vaccines, are a promising therapeutic option for many diseases as they offer the customization of their ability on protection and treatment with high stability. The production of DNA vaccines is considered rapid and less complicated compared to others such as mRNA vaccines, viral vaccines, or subunit protein vaccines. However, the main issue for DNA vaccines is how to produce the active DNA, a supercoiled isoform, to comply with the regulations. Our work therefore focuses on gaining a process understanding of the purification step which processes parameters that have impacts on the critical quality attribute (CQA), supercoiled DNA and performance attribute (PA), and step yield. Herein, pVax1/lacZ was used as a model. The process parameters of interest were sample application flow rates and salt concentration at washing step and at elution step in the hydrophobic interaction chromatography (HIC). Using a Design of Experiment (DoE) with central composite face centered (CCF) approach, 14 experiments plus four additional runs at the center points were created. The response data was used to establish regression predictive models and simulation was conducted in 10,000 runs to provide tolerance intervals of these CQA and PA. The approach of this process understanding can be applied for Quality by Design (QbD) on other DNA vaccines and on a larger production scale as well.

Plasmid DNA is a vital biological tool for molecular cloning and transgene expression of recombinant proteins; however, decades ago, it has become an exceptionally appealing as a potential biopharmaceutical product as genetic immunization for animal and human use. The demand for large-quantity production of DNA vaccines also increases. Thus, we, herein, presented a systematic approach for process characterization of fedbatch Escherichia coli DH5α fermentation producing a porcine DNA vaccine. Design of Experiments (DoE) was employed to determine process parameters that have impacts on a critical quality attribute of the product, which is the active form of plasmid DNA referred as supercoiled plasmid DNA content, as well as the performance attributes, which are volumetric yield and specific yield from fermentation. The parameters of interest were temperature, pH, dissolved oxygen, cultivation time, and feed rate. Using the definitive-screening design, there were 16 runs, including 3 additional center points to create the predictive model, which then was used to simulate the operational ranges for capability analysis.

Abstract:The production of biopharmaceutical in lab scale differs from in pilot plant-scale particularly in process control or conditions control aspects. For larger volume of production, there is a chance of having deviation on controlling the operating conditions that is likely to have effects on specifications of biopharmaceuticals. Thus, this work focused on the optimization and robustness study of growth conditions on microbial fermentation using design of experiment methodology (DoE) and statistical analysis approach. Three factors for designing experiment were temperature, pH and % dissolved oxygen (DO). A high-throughput MRT-24 microbioreactor and Pichia pastoris KM71H which expresses Japanese Encephalitis virus envelope protein were used as a model study and the measured output was OD600. After 17 hours of fermentation, the data was analyzed and the conditions where temperature was 28.0 °C, pH was 6.37 and DO was 30% were calculated to be the optimized value. Then the robustness study was carried out in which 5 % loss of the highest yield was set as a criterion. The results predicted that the robust range was temperature of 28:Cto 29.5°C and pH of 6.0 to 6.5.

Porcine Circovirus type 2d (PCV2d) is becoming the predominant PCV genotype and considerably affects the global pig industry. Nevertheless, currently no commercial PCV2d vaccine is available. Preventing and controlling the disease caused by PCV2d is therefore based on other genotype-based vaccines. However, their production platforms are laborious, limited in expression level and relatively expensive for veterinary applications. To address these challenges, we have developed a simple and cost-efficient platform for a novel PCV2d vaccine production platform, using fed-batch E. coli fermentation followed by cell disruption and filtration, and a single purification step via cation exchange chromatography. The process was developed at bench scale and then pilot scale where the PCV2d subunit protein yield was approximately 0.8 g/L fermentation volume in a short production time. Moreover, we have successfully implemented this production process at two different sites, in Southeast Asia and Europe. This demonstrates transferability and the high potential for successful industrial production.